Listeria is a genus of ubiquitous bacteria that are gram-positive and non-sporulating and consist essentially of the species Listeria monocytogenes, L. innocua, L. welshimeri, L. seeligeri and L. ivanovii and L grayi. Among these, only some strains of the species L. monocytogenes are food borne pathogens for humans, in particular to those with a weakened immune system and for the elderly and the newborn. The most common symptoms of listeriosis are septicemia, meningitis, and miscarriages.
A large number of methods for detecting Listeria monocytogenes are known. Conventional detection methods for L. monocytogenes require culture enrichment steps to increase the number of Listeria cells to a detectable level. After culture enrichment the cells are then allowed to grow out on specific nutrient agarose plates forming individual colonies with distinct morphology allowing for their isolation (Lovett et al., J. Food Protection 50 (1987), 188-192; McClain & Lee, J. Assoc. Off. Anal. Chem. 71 (1988), 660-664). Single colonies are examined for their morphology, biochemical and serological properties. An analysis may take up to 6-8 days to confirm the presence of Listeria. Consequently, rapid detection processes for detecting Listeria, in particular in foodstuffs or clinical and environmental samples are urgently required.
Various high-speed methods for detecting Listeria monocytogenes have been developed. Such methods are based either on immunological methods, the use of polymerase chain reaction (PCR) technology, or on the application of nucleic acid probes.
Some test kits for detection of Listeria monocytogenes by means of antibodies are already commercially available. Most of these tests however require at least 10,000 cells for detection. While the immunological tests that are currently on the market only take a few hours, they require a lengthy culture enrichment step(s).
Detection of Listeria monocytogenes may be carried out by direct hybridization of probes to microbe-specific DNA or RNA (for example, Datta, A. R. et al., Appl. Environ. Microbiol. 53 (1987), 2256-2259). The major disadvantage of such methods is the low sensitivity, since at least 105-106 copies of the target nucleic acid are required. This can be compensated for by the amplification of the target sequence, for example using the polymerase chain reaction (PCR). A plurality of PCR methods for detecting L. monocytogenes has been described in the literature [for a review see, for example, Jones, D. D. & Bej, A. K. in “PCR Technology, Current Innovations”, Griffin, H. G & Griffin, A. M., eds., (1994), 341-365]. See also U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188. Furthermore, the ligase chain reaction [WO publication 89/09835], “self-sustained sequence replication” [EP 329,822], “transcription based amplification system” [EP 310, 229], and Qβ RNA replicase system [U.S. Pat. No. 4,957,858] may be employed for the amplification of nucleic acids.
PCR permits the in vitro amplification of targeted nucleic acids. This increases the sensitivity of detection to fewer cells and subsequently can reduce the length of time needed for culture enrichment. To start the reaction, short nucleic acid fragments (primers) are required. Primers function as pairs with each set encompassing the section of the genome that is to be amplified. Both of the primers are the complementary sequence to the relevant section of the target gene sequence. Since each primer is a complementary sequence it can hybridize with one nucleic acid strand. The formation of this hybridization allows for the enzyme DNA polymerase to direct the synthesis of a complementary strand that is an extension of the primer. Temperature regulated hybridization cycling and the use of thermal stable DNA polymerases are the basis for PCR directed nucleic acid amplification. The choice of the primer pairs determines the specificity of the detection reaction. The use of this process for detecting L. monocytogenes is described in Appl. Environmental Microbiology 57, 606-609 (1991), in Letters Appl. Microbiol. 11, 158-162 (1990) and in J. Appl. Bact. 70, 372-379 (1991). More extensive information regarding the details of these processes is available in these publications, PCR Primer A Laboratory Manual CSHL Press(1995) Diffenbach, C. W. and Dveksler, G. S. and Real Time PCR An Essential Guide Horizon Biosciences (2004) Edwards K, Logan, J and Sander, N.
The detection methods described for L. monocytogenes are based mainly on targeting genes that play a role in the pathogenicity of L. monocytogenes. It is known that some of these genes are located on the chromosome next to each other in a virulence gene cluster (Pathogenicity Islands and Other Mobile Virulence Elements, ASM Press (1999) Kaper, J. B. and Hacker, J.). Since the listeriolysin gene (hlyA) has been recognized as a necessary gene for the pathogenicity of L. monocytogenes (Cossart, P. et al., Infect. Immun. 57 (1989), 3629-3636), most of the genotypic detection methods are based on this gene sequence.
Although strains of Listeria monocytogenes are the only pathogens to humans, testing for this specific group would be limiting in an effort to identify potential growth habitats for them in food manufacturing facilities. Thus, testing to detect all Listeria species is useful in the identification of harborage site. This identification is necessary to allow for through sanitation and the subsequent elimination of Listeria. 
Detection of groups of bacteria within a given genus requires the identification of gene(s), which have conserved sequences among the target group. Additionally the conserved sequences need to be unique compared to all other non-Listeria bacteria to allow for a discriminating test. Genes that have been used for this purpose have typically been of ribosomal origin (J. of Food Protection 1995 58(8) 867-873) or derived from hly (WO9844153) and iap (Applied and Environ Micro 1992 58(8) 2625-2632). The draw-back to ribosomal genes is that they have different copy numbers for different species making quantitation difficult for a genus specific test. The other genes mentioned may have similarity to other sequences that are not of Listeria origin, thereby decreasing test specificity. The greater the test specificity of the assay determines which culture enrichment media is appropriate. The use of a non-selective culture enrichment can be employed with a Listeria specific DNA assay, since non-Listeria would be discriminated for by targeting a unique Listeria DNA fragment. Use of selective media can slow the growth and inhibit the recovery of injured Listeria requiring a longer incubation period to reach a detectable cell number that relates to an equivalent target DNA copy number. While a non-selective medium can allow for unimpeded optimal growth, ideally single copy genes that have conserved sequence homology within the target Listeria genus and span a length of between 100 to 400 base pairs make excellent diagnostic markers.
In view of the above, there is a need for oligonucleotides that can be utilized for diagnostic purposes to detect low levels of the Listeria genus. These oligonucleotides can be applied to the many different PCR amplification techniques to provide a quick, sensitive and specific test. The sensitivity and specificity of such a test will make it possible to reduce the incubation time of a culture enrichment step thereby decreasing the overall time needed to get a test result.